Impaired TLR2 and TLR7 response in olive flounder infected with viral haemorrhagic septicaemia virus at host susceptible 15 °C but high at non-susceptible 20 °C.

The olive flounder (Paralichthys olivaceus) is susceptible to viral haemorrhagic septicaemia virus (VHSV) at 15 °C but no mortality is observed at 20 °C even though the virus can grow profusely in vitro. Thus, we designed an experiment to better understand the immune response of olive flounder to VHSV when the host reared at 15 °C or 20 °C and infected with the virus. Olive flounder (18-22 g) reared at 15 ± 0.5 °C or 20 ± 0.5 °C were intra-peritoneally injected with VHSV (10(7.8) TCID50/fish) and sampled (n = 5) for head kidney at 3, 6, 12 hpi, 1, 2, 4 and 7 dpi; similarly, mock injected control groups (n = 5). Real-time PCR-based absolute quantification method was followed to quantify copies of VHSV gRNA and mRNA, while the immune gene expression of the olive flounder was quantified relative to internal control, β-actin. Viral infection resulted in a cumulative mortality of 24% in olive flounder reared at 15 °C, but no mortality was recorded in the 20 °C group or control groups. TLR2 and TLR7 expression at 15 °C was enhanced during early-infection phase (3-6 hpi) and recovery phase (4-7 dpi) when viral transcription was low, but expression was significantly reduced (12 hpi-1 dpi) at peak-infection period. However, the 20 °C group showed low viral transcription and expressed high level of TLR7 and a moderately higher unchanged level of TLR2. In both the groups, TLR3 expression was unaffected. Nevertheless, expression of MDA5 and LGP2 increased significantly irrespective of rearing temperature at the time of peak infection, hence at 15 °C VHSV down-regulated expression of TLR2 and TLR7 but not MDA5 or LGP2. Comparatively, at 15 °C IRF3 expressed high but IRF7 remained very low. Interleukins (IL-1β, IL-6 and IL-8) were significantly elevated in both the groups, but quicker and for a shorter period at 20 °C. In the 15 °C group, an extended period of expression of ILs could create an unsafe prolonged inflammatory condition. The olive flounders expressed high ISGs at 15 °C but were lagging by 12 h than 20 °C group. Based on these findings, we concluded that viral-mediated disruption of TLR2 and TLR7 expression in the 15 °C group could have delayed the host interferon response and provided a window for high viral growth. However, an effective host immune response at 20 °C contained VHSV from reaching the critical limit.